PROJECT SUMMARY/ABSTRACT The goal of this proposal is to understand the upstream molecular mechanisms underlying the spread of tau- mediated neurofibrillary tangle pathology within the highly integrated cholinergic nucleus basalis-default mode network (Ch4-DMN) connectome during the transition from no cognitive impairment (NCI) to mild cognitive impairment (MCI). We recently showed that toxic tau oligomer accumulation within Ch4 subfields follows a caudal-to-rostral gradient during disease progression. Our new pilot data show that tau oligomer accumulation within DMN hubs begins in the precuneus (PreC), which is innervated by the caudal Ch4 subfields, before spreading to the frontal cortex (FC), which is innervated by the rostral Ch4 subfields. Hence, the spatiotemporal pattern of tangle evolution within the DMN may mirror the topography of tau pathology observed in innervating Ch4 subfields, suggesting a pathological spread of neurodegeneration within Ch4-DMN circuits. Aim 1 will test this hypothesis by interrogating DMN tissue categorized as low pathology (LP)-NCI, high pathology (HP)-NCI, MCI, or Alzheimer?s disease (AD) with site-specific tau pretangle antibodies, unbiased stereology, and optical density methods. The molecular mechanisms driving tau pathology within the Ch4-DMN connectome are unknown and may provide key insights into novel disease-modifying targets. Our previous gene expression profiling of Ch4 neurons revealed alterations in tau metabolic pathways in MCI, including increased expression of tau kinases, decreased expression of tau phosphatases, and skewing of the ratio of 3-repeat to 4-repeat tau isoforms. One potential regulatory pathway causing these changes may involve small non-coding microRNAs (miRNAs), which control mRNA stability. Pilot miRNA sequencing revealed multiple miRNAs dysregulated in MCI and AD that target tau metabolism. In particular, miR-298 was significantly upregulated in AD, whereas miR- 298 overexpression in human mixed brain cultures reduced the levels of higher molecular-weight tau monomers, suggesting miR-298 influences tau isoform composition. Therefore, Aim 2 will test the hypothesis that miRNAs targeting tau metabolic mRNAs are dysregulated within the DMN connectome in HP-NCI and MCI by using laser capture microdissection of pretangle-bearing DMN cortical neurons, followed by miRNA sequencing, bioinformatics, target mRNA analysis, and validation studies. Aim 3 will then use a host of stringent miRNA- mRNA interaction and expression assays, tau biochemical, and neuronal morphometric and functional analyses to test the hypothesis that candidate miRNAs identified in Aim 2 will mechanistically interact with tau and tau metabolic pathway mRNAs to impact tau pathology in human mixed brain cultures. We will also explore miRNAs related to cholinergic, RNA splicing, synaptic, amyloid, and inflammatory pathways in collaboration with Projects 3 and 4. Altogether, these project aims will identify select miRNA pathways as critical upstream regulators of tau metabolism related to the pathological aggregation and circuit-based spread of tau pathology within the Ch4- DMN connectome prior to MCI, thus revealing novel potential targets for therapy.